CN106377540A - Dual-action compounds targeting adenosine A2A receptor and adenosine transporter for prevention and treatment of neurodegenerative diseases - Google Patents

Abstract

The present invention provides therapeutic agents for preventing and treating neurodegenerative diseases. These agents concomittantly target both the adenosine A2A receptor (A2AR) and the equilibrative nucleoside transporter 1 (ENT1).

Description

It is bound to adenosine A2AAcceptor and adenosine transport are to prevent and to treat neurodegenerative disease Dual-function compound

The application is that (PCT international application no is for Application No. 201080051296.X submitted on November 12nd, 2010 PCT/US2010/056516), invention entitled it is bound to adenosine A_2AAcceptor and adenosine transport are to prevent and to treat nerve degeneration The divisional application of the application for a patent for invention of the dual-function compound of disease.

The application request enjoys in the preferential of the U.S. Provisional Patent Application 61/260,932 that on November 13rd, 2009 submits Power, and quoted in full herein by reference as.

Technical field

The present invention provides one kind in order to treat neurodegenerative disease (neurodegenerative diseases), such as prosperous fourth The identification of compound, synthesis and the use of chorea (Huntington ' s disease, HD).

Background technology

Huntington's disease (HD) is a kind of because of Huntington gene (Huntingtin gene, Htt) upper CAG tri- even nucleosides The Autosome dominant neurologic degenerative disease (autosomal that acid extends (CAG trinucleotide expansion) and causes Dominant neurodegenerative disease), this disease can show chorea (chorea), lose intelligence And mental symptom (psychiatric symptoms) (dementia).^1-3Although existing part has the medicine of gentle symptom effect It is published⁴ _, ⁵, but for Huntington's disease effectively treatment method still have yet-to-be developed.It is currently known to turn in gene and grow mouse mould In formula experiment, selective A_2AExcitant (the selective A of adenosine receptor_2Aadenosine receptor(A_2AR) excited Agent), CGS21680 (abbreviation CGS), the symptom of Huntington's disease of can releiving⁶, and, this compound has been found to permissible Solved in Huntington's disease by the activity strengthening ubiquitin protein enzyme system (ubiquitin-proteasome system) The problem of urea cycle defect⁷.However, it is known that CGS more can cause immunosuppressive action in addition to existing side effect⁸, therefore It is not suitable for utilization clinically.On the other hand, one kind separate from rhizoma Gastrodiae (Gastrodia elata Bl) and It is named as the neplanocin of T1-11 (compound 1), be also a kind of A_2AThe excitant of adenosine receptor, has been shown to have suppression The machine of the serum deprivation institute trigger cell apoptosis of PC12 cell turn (serum-deprived apoptosis) effect it is thus possible to There are the potentiality for the treatment of neurodegenerative disease⁹.

Abbreviation：

A_2AR：A_2AAdenosine receptor.

Ac₂O：Acetic anhydride (acetic anhydride).

CGS：6- amido -9- (5- ethylaminocarbonyl -3,4- dihydroxy-oxolan -2- base) -2- { 2- [4- (2- carboxylic Ethyl) phenyl] ethamine } purine (6-amino-9- (5-ethylcarbamoyl-3,4-dihydroxy-oxolan-2-yl) -2- {2-[4-(2-carboxyethyl)phenyl]ethylamino}purine).

DIEA：Diisopropylethylamine (diisopropylethylamine).

DMF：N,N-dimethylformamide (N, N-dimethylformamide).

DML：Many combinations target part (designed multiple ligands) of design.

ENT：Balanced type nucleoside transporting (equilibrative nucleotide transporter).

ESI：EFI spills ionization (eletrospray ionization).

EtOAc：Ethyl acetate (ethyl acetate).

HD：Huntington's disease (Huntington ' s disease).

hENT1：Mankind's balanced type nucleoside transporting 1 (human equilibrative nucleotide transporter 1).

HBA：Hydrogen bond receptor (hydrogen bond acceptor).

HBD：Hydrogen-bond donor (hydrogen bond donor).

HBTU：2- (1H- BTA -1- base) -1,1,3,3- tetramethylurea (2- (1H-benzotriazol-1-yl) - 1,1,3,3-tetramethyluronium).

HOBt：1- hydroxyl BTA (1-hydroxybenzotriazole).

HP：Water repellent region (hydrophobic).

HPLC：High-effect LC (high-performance liquid chromatography).

IR：Infrared spectrum (infrared).

JNK：C-Jun N- terminal Kinase (c-Jun N-terminal kinase).

MS：Mass spectrometry (mass spectrometry).

MsCl：Mesyl chloride (methanesulfonyl chloride).

MTT：3- (4,5- dimethylthiazole -2- base) -2,5- diphenol tetrazolium bromide ((3- (4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)).

NBTI：S- (4- nitre benzyl) -6- thioinosine (S- (4-nitrobenzyl) -6-thionosine)).

NMR：Nuclear magnetic resonance (nuclear magnetic resonance).

py：Pyridine (pyridine).

PyBOP：BTA -1- base-epoxide tripyrrole alkyl phosphorus hexafluorophosphoric acid (benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate).

RA：Aromatic rings (ring aromatic).

t-BuOH：Tertiary butyl alcohol (tertiary butanol).

TEMPO：2,2,6,6- tetramethyl piperidine -1- epoxide (2,2,6,6-tetramethylpiperidinyl-1- oxy).

THF：Oxolane (tetrahydrofuran).

TLC：Thin-layer chromatography (thin-layer chromatography).

TsCl：Paratoluensulfonyl chloride (p-toluenesulfonyl chloride).

TsOH：P-methyl benzenesulfonic acid (p-toluenesulfonic acid).

ZM：4- (2- [7- amido -2- { 2- furyl } { 1,2,4 } triazolone { 2,3-a } { 1,3,5 } triazine -5- base-amine Base] ethyl) phenol (4- (2- [7-Amino-2- { 2-furyl } { 1,2,4 } triazolo { 2,3-a } { 1,3,5 } triazin-5- yl-amino]ethyl)phenol).

Content of the invention

A_2AR and adenosine transport (adenosine transporter, for example, balanced type nucleoside transporting is sub (ENT1)) are all Have been found to be positioned at corpus straitum (striatum)^10–13, that is, the place of the Huntington gene gathering being mutated¹⁴.Suppression adenosine turns Fortune can regionally improve the concentration of adenosine, and and then lifts activation (agonizing) A_2AThe efficiency of R.It is interesting that chemical combination Thing 1 is also considered as a kind of ENT1 inhibitor.On the other hand, the potential immunosuppressive action of CGS is also A_2AR message passes The result passed, display and A_2AR has the advantageous feature that very strong binding affinity may not be clinically active drug.

Therefore many researchs are devoted to modified compound 1 but retain its multi-functional characteristic.Additionally, for the foregoing reasons, The compound that the compound of the of short duration or faint binding ability of tool may relatively have stable bond ability is more suitable for¹⁵, especially in knot Together in A_2AIn the case of R massage transfer system.Combine target part (designed multiple obtained by design more Ligands, DMLs)^16,17Be entirely different concept through mixed and disorderly compound obtained by random screening because they are through bright Really design and obtain the characteristic optimization so as to be presented.Compared to the single combination target compound (individually- of tool Targeting compounds), scientific circles think that DMLs may have the disease of the complicated cause of disease in treatment, such as pant (asthma), on obesity (obesity), cancer (cancer) and mental illness, there is advantage^16-18.It is generally understood that organism The repeatability of interior complicated contact network possibly causes high selectivity medicine cannot show expected therapeutic effect with viability The reason¹⁹.Recently assessment display A_2AThe pharmacological property of R is really considerably complicated, and therefore, DMLs may be particularly suited for combining again To A_2AR massage transfer system²⁰.However, one because being difficult to, by computer program, correctly framework goes out a part and numerous marks Interactive relation, two come the many restrictions in view of chemical synthesis and the requirement of the physicochemical characteristic to compound, therefore The design target part that combines is often an extremely challenging task more^21,22.Perhaps it is multiple to be because more combination targets Miscellaneous degree is higher, thus at present it is reported that DMLs most be double function ligand (dual-function ligand)²².

In our previous U.S. Provisional Application case (U.S. Provisional Application cases number：61/260,932) multiple ECDCs are mentioned in Become and to adenosine receptor and transhipment, there is bifunctional compound.These compounds have an adenosine structural bone in general Frame, and in its N⁶And C^5’Position carries the compound of the structure of following modification：

Wherein, n is 1 to 3；R¹It is selected from (being substituted) benzene ((substituted)-benzene), aromatic hydrocarbons And the group that formed of heterocycle (heterocycle) (polyarene)；R²It is selected from halogen, hydroxyl, alkoxyl, repeatedly nitrogen base (azido), amido, (being substituted) amido, amide groups, sulfydryl (sulfanyl), sulfonyl (sulfonyl), triazolyl and cyanogen The group that base is formed；And R³It is selected from (being substituted) carbonyl, carboxylic acid；(being substituted) urea groups ((substituted) Carbamide), the group that cyano group, (being substituted) alkynyl and (being substituted) tetrazole radical are formed.

Preferably, in one embodiment of the invention, this compound has having structure：

Wherein, the heterocycle that this is substituted contains 5 yuan or 6 yuan of rings and is somebody's turn to do and closes heterocycle (fused heterocycle) and contain Nitrogen, oxygen or sulphur are as hetero atom, and this substituent is (containing 1 to 6 selected from hydrogen, halogen (fluorine, chlorine, bromine and iodine), hydroxyl, alkyl Individual carbon), the group that formed of trifluoromethyl (trifluoromethyl) and (being substituted) phenyl group.

This heterocycle can be pyrroles, furans, thiophene, pyridine, hexahydropyridine (piperidine), hexahydro pyrrole (piperazine), indoles, benzofuran (benzofuran), benzothiophene or quinoline.

In a further preferred embodiment, this compound has having structure：

Wherein, this aromatic rings is the group being constituted selected from benzene, naphthalene, anthracene, phenanthrene and pyrene；Work as n=1, this benzyl group (benzyl group) optionally has selected from halogen (fluorine, chlorine, bromine and iodine), alkyl (methyl, ethyl, propyl group, fourth Base and trifluoromethyl), phenyl (phenyl), carboxyl, alkoxyl (OR, wherein R=CH₃、C₂H₅、C₃H₇Or C₄H₉), amido (NRR ', wherein R and R ' represent H, CH₃、C₂H₅、C₃H₇、C₄H₉Or phenyl), amide groups (NHCOR, wherein R=CH₃、C₂H₅、C₃H₇ Or C₄H₉), nitro, sulfonate (sulfonate), alkanoyl (COR, wherein R=H, CH₃、C₂H₅、C₃H₇、C₄H₉Or phenyl) and carboxylic Hydrochlorate (CO₂R, wherein R=H, CH₃、C₂H₅、C₃H₇、C₄H₉Or phenyl) substituent of group that constituted.

Preferably, this compound has having structure：

Wherein, R³The group being constituted selected from hydrogen, alkyl (containing 1 to 4 carbon) and (being substituted) phenyl, and R¹As Upper described.

Another preferred compound has having structure：

Wherein, R⁴Be selected from hydrogen, halogen (fluorine, chlorine, bromine and iodine), hydroxyl, alkyl (containing 1 to 6 carbon), trifluoromethyl and The group that (being substituted) phenyl is constituted, and R¹As mentioned above.

Further preferably compound has having structure：

Wherein, R⁵It is the group being constituted selected from hydrogen and alkyl (containing 1 to 4 carbon), and R¹As mentioned above.

Another preferred compound has having structure：

Wherein, R is selected from hydrogen, halogen (fluorine, chlorine, bromine and iodine), alkyl (containing 1 to 4 carbon), trifluoromethyl, phenyl, hydroxyl Base, alkoxyl (containing 1 to 4 carbon), (being substituted) amido, (being substituted) amide groups, nitro, sulfonate, carbonyl and carboxylic acid The group that salt is constituted, and it is in ortho position, meta or para position；Wherein, x is 1 to 5.

Another embodiment of the invention provides a kind for the treatment of method of neurodegenerative disease, and it comprises：By effective dose At least one above-claimed cpd bestow an object in need for the treatment of.This neurodegenerative disease can refer to because of protein mistake folding institute The protein mistake folding disease causing.This protein mistake folding disease includes Alzheimer's disease (Alzheimer ' s Disease), Parkinson's disease (Parkinson ' s disease), ALS (spinocerebellar ataxia Disease), Puli hold high disease (Prion disease), Huntington's disease and spinocerebellar ataxia disease (amyotrophic lateral sclerosis).The method of the present invention is particularly useful for treatment Huntington's disease.

Another embodiment of the invention provides a kind of composition, and it comprises at least one above-claimed cpd of effective dose And medicine acceptable carrier.

In order to reasonable design is suitable for the double function ligand of the healing potion as Huntington's disease, our construction first Two pharmacophore models (pharmacophore model), one of them is A_2AR excitant (A_2AR excitant), another Individual, it is ENT1 inhibitor.Three-dimensional pharmacophoric group can assume a series of active region of the identical biomolecule of combinations and represent The particular spatial distribution of the chemical functional characteristic of the compound of identical function²³, the structure of this biomolecule in this combination there is no When method is solved with high-resolution X-ray or NMR, particularly useful.Pharmacophore analysis have successfully applied to multi-medicament R＆D work^24-29.Although human adenosine A_2AThe high parsing crystal structure of acceptor has solved³⁰, but it is shown that itself and antagonism Agent, the configuration that ZM241385 (abbreviation ZM) combines, rather than excitant.On the other hand, the crystal structure of mankind ENT1 is then not yet bright Bright, also fail at present find suitable structure as template to carry out homology modeling (homology modeling).

We devise a series of derivative of adenosines (Fig. 1) based on the structural framework of compound 1.As long as pharmacophoric group is fitted Property analysis (pharmacophore fitting) predict that this modified compound has acceptable activity, this chemical modification It is used.Additionally, and using Ligand Competition binding tests (competitive ligand binding assay) to determine The compound of this design is really and A_2AR and ENT1 has good binding affinity.Finally, these compounds are through testing further Whether they can avoid the PC12 cell of serum deprivation culture to start apoptosis machine turns, and this whether will be them have treatment nerve The key characteristics of the potentiality of degenerative disease^31,32.

Brief description

Fig. 1 display CGS, NBTI and other are through being designed at N⁶- and C^5′The structure of the adenosine derivative that-position is modified.

Fig. 2 shows adenosine A_2AThe three-dimensional pharmacophore model of receptor agonist；(A) architectural feature of pharmacophore model, Blue：Water repellent region (HP), golden：Aromatic rings (RA), red：Hydrogen-bond donor (HBD), green：Hydrogen bond receptor (HBA)；(B) CGS and the identical situation of pharmacophore model；(C) the identical situation of compound 1 and pharmacophore model；(D) NBTI's is identical Situation；(E) the identical situation of compound 6；(F) the identical situation of compound 11.

Fig. 3 shows the three-dimensional pharmacophore model of mankind's balanced type nucleoside transporting (hENT1) inhibitor；(A) drug effect base The architectural feature of group's model, golden：Aromatic rings (RA), green：Hydrogen bond receptor (HBA；The distance of HBA1 and RA is The distance of HBA2 and RA is)；(B) the identical situation of NBTI and pharmacophore model；(C) the identical shape of compound 1 Condition；(D) the identical situation of CGS；(E) the identical situation of compound 6；(F) the identical situation of compound 11.

Fig. 4 is a scatter diagram, its corresponding display A_2A- R anti-depressant pKi predicted value and measured value；Wherein solid circles table Show the compound of training data, and empty circles represent the compound of synthesis.

Fig. 5 is a scatter diagram, and it corresponds to pKi predicted value of display ENT1 inhibitor and measured value；Wherein solid circles table Show the compound of training data, and empty circles represent the compound of synthesis.

Specific embodiment

Human adenosine A_2AThe pharmacophore model of receptor agonist

In double pharmacophoric group drug design programs, mankind A_2AR(hA_2AR) anti-depressant three-dimensional pharmacophore model first It is established and can be provided as hA for design_2AThe anti-depressant compound of R.Training data (the training selecting in this specification Set 25 different degrees of structure variations of tool and hA) are included_2AThe chemical combination of R activity (187 μM of Ki from 1.2 nM to) Thing.Has potential hA_2AR excitant, CGS³³, it is also included within this training data.RecycleCompany'sModule³⁴Carry out the pharmacophore model of these parts of construction.This construction pharmacophoric group out is As shown in Figure 2 A, it shows four architectural features, including：Water repellent region (hydrophobic, HP are shown as cyan), virtue Fragrant ring (ring aromatic is shown as golden), hydrogen-bond donor (hydrogen bond donor, HBD, shown in red) and Hydrogen bond receptor (hydrogen bond acceptor, HBA, shown in green).Four features of the pharmacophoric group of CGS and construction All can be good coincide (Fig. 2 B).Relatively, S- (4- nitre benzyl) -6- thioinosine (S- (4-nitrobenzyl) -6- Thioinosine, NBTI)³⁵Although presenting the adhesion with the sub- strength of adenosine transport, in default of the kiss of an aromatic rings Close (Fig. 2 D) and as hA_2ADuring R part, only has weak affinity.The double function ligand 1,6 and 11 of this design is then at least fit to A_2AThree features (Fig. 2 C, Fig. 2 E and Fig. 2 F) of the anti-depressant pharmacophore model of R.

The pharmacophore model of the inhibitor of balanced type nucleoside transporting

In order to design one at the same time as hA_2AR excitant and the dual-function compound of hENT1 inhibitor, hENT1 suppresses The pharmacophore model of agent is similarly constructed (Fig. 3 A).The training data selected in this specification includes 25 and has difference The compound of the hENT1 inhibitory activity of degree, its IC₅₀It is between 0.29nM-32 μM.The medicine of the hENT1 inhibitor of this construction Effect group model is only made up of three features, including two hydrogen bond receptors and an aromatic rings.This five compounds (NBTI, 1, CGS, 6 and 11) these three features of all can coincideing.In hA_2AThe pharmacophoric group of the anti-depressant pharmacophoric group of R and hENT1 inhibitor it Between the identical characteristic reaction of varying number go out the middle compound characteristic of itself.

Careful inspects as hA_2AIt has been found that majorityization after the structure of compound in R anti-depressant test data group Compound, is especially identified the compound with higher potentiality, including CGS, carries a hydrophobicity base in 5 ' positions of nucleosides Group.Therefore, the pharmacophoric group of construction is set and must include this important feature.Contrary, as hENT1 inhibitor Test data set in, 5 ' positions of the nucleosides of nearly all compound all carry the hydroxyl groups of a polarity.

Two differences observed between target pharmacophoric group also provide the inspiration of design dual-function compound.Citing comes Say, compound 6 can not fit like a glove A_2AThe hydrophobic character of the pharmacophore model of acceptor, if therefore it obviously can not coincide The CGS of all features is potential.However, compound 6 but can be good with three features in hENT1 model coincide.

As for compound 11, it can be coincide with all of feature, including A_2AThe hydrophobic character of R pharmacophore model, But it is still equally well fit to hENT1 pharmacophore model, display ENT1 pharmacophore model has higher tolerance (tolerance).In summary, the degree coincideing by the feature comparing this two pharmacophore models and compound, we Assume that this hA_2AThere is in the binding pocket (binding pocket) of R an important water repellent region, and this hENT1 Hydrophobic parts (the hydrophobic of the 5 ' positions possibly for the nucleosides accommodating this series of compound for the binding pocket Moieties) more elastic.In the case of lacking the structural information of hENT1, pharmacophore analysis provide us in design And required information on understanding dual-function compound.

The synthesis of adenosine derivative

The representational neplanocin of a group is to set up (reaction process 1) according to this pharmacophore model.It is certainly earliest Rhizoma Gastrodiae⁹The compound 1 separated can high productivity by making 6-chloropurine core furanoside (6-chloropurine Ribofuranoside) (17) (4-hydroxybenzylamine, with hydrogen chloride salt with 4-Hydrobenzylamine The form of (hydrochloric salt)) enter under the alkaline environment of diisopropylethylamine (diisopropylethylamine) Row substitution reaction and obtain.Because 4-Hydrobenzylamine cannot be obtained with commercial system, therefore to be prepared with two steps：Make 4- Hydroxy benzaldehyde generates monoxime with hydroxylamine (hydroxylamine) by condensation reaction (condensation reaction) Class (oxime) intermediate product；Then this oximes intermediate product is made to carry out hydrogenation under the catalysis of Pd/C and HCl.Similar to The preparation procedure of compound 1, by 6-chloropurine core furanoside and the benzene methanamine being suitably substituted carry out substitution reaction with A series of Ns are obtained⁶The adenosine derivative 2-6 that position is substituted.In N⁶- (3- indolylethyl) adenosine (N⁶-(3-indolyethyl) Adenosine) in the preparation of (6), traditional heating means are replaced using microwave, to shorten the reaction time.

N is made in anhydrous propanone⁶- (4- methoxybenzyl) adenosine 3 and 2,2- dimethoxy propane (2,2- Dimethoxypropane) acetone acetalation derivative (3-acetonide) of raw the corresponding compound 3 of reaction, it is then React in the presence of pyridine (pyridine) with paratoluensulfonyl chloride (p-toluenesulfonyl chloride) and obtain one Mixture, it contains the toluenesulfonic acid of above-mentioned 3-acetonide (tosylate) derivative and chlorine derivative (reaction process 1). This toluenesulfonic acid derivative is simultaneously unstable, conversely, this chlorine compound (acetone acetalation derivative (7- of compound 7 Acetonide)) be then able to by chromatography separate, then continue hydrolysis and obtain compound 7.Said mixture is not being entered one React with sodium azide (sodium azide) in the case of step is detached, and and then through acid-catalyzed hydrolysis, tool repeatedly nitrogen can be generated The compound 8 (azido compound 8) of base.In addition, making acetone acetalation derivative (8-acetonide) of compound 8 enter Row staudinger reaction (Staudinger reduction) can generate an amine intermediate product, and this amine intermediate product is in acid Corresponding acetamide compound 9 is can be exchanged into after removing its 2 ', 3 '-isopropylidene group (2 ', 3 '-isopropylidene) (acetamide 9) and sulfonamide compounds 10 (sulfonamide 10).

Reaction process 1, N⁶- (4- methoxybenzyl) adenosine (N⁶- (4-methoxybenzyl) adenosine) (3) conjunction Become and its its 5 ' hydroxyl is substituted by chlorine, repeatedly nitrogen base (azide), acetamide (acetamide) and para toluene sulfonamide (p- respectively Toluenesulfonamide reaction process).

Reaction process 2, is obtained by the repeatedly nitrogen base group that quick-acting closes reaction (click reaction) modification 5 ' positions Triazole compounds (triazole compound).

In another reaction process (reaction process 2), make compound 8 and 1- hexin, 1- octyne and the 3- of tool repeatedly nitrogen base Phenyl -1- propine carries out Cu⁺(1,3-dipolar cycloaddition, quick-acting closes 1, the 3- Dipolar Cycloaddition of catalysis Reaction)³⁸Triazole derivative 12,13 and 14 can be obtained.Similarly, make the acetone acetalation derivative (8- of compound 8 Acetonide) carry out quick-acting conjunction reaction with propargyl alcohol (propargyl alcohol) and triazole compounds can be generated, this three The hydroxyl of azole compounds is activated during the course of the reaction and becomes a methanesulfonic acid (mesylate), and then by NaBH₄It is reduced to Monomethyl.After again 2 ', 3 '-isopropylidene group being removed, you can obtain compound 11.

Reaction process 3, carboxamides derivatives (carboxamide derivatives) and terazole derivatives (tetrazole Derivatives synthesis).

Acetone acetalation derivative (acetonide) making 6-chloropurine core furanoside is in 2,2,6,6- tetramethyl piperazine Pyridine -1- epoxide (2,2,6,6-tetramethylpiperidinyl-1-oxy, TEMPO)³⁹Catalysis under by iodobenzene diacetate ((diacetoxyiodo) benzene) oxidation generates monocarboxylic acid compound 18 (carboxylic acid 18).Make this Carboxylation Compound 18 carries out even summation reaction and generates amide compound 19 and 20 (amide 19, amide 20) respectively with ethamine and ammoniacal liquor. So that the chlorine atom of this compound 19 is replaced by 4-Hydrobenzylamine and after acetone acetal is hydrolyzed, that is, amide compound 15 is obtained (amide 15).On the other hand, amide compound 20 and Me₂SO, oxalyl group (oxalyl), chlorine and diisopropyl ethyl amine (i- Pr₂Net be then converted to nitrile compound 21 (nitrile 21) after) reacting⁴⁰.In chlorine atom, it is replaced by 4- methoxybenzyl amine Afterwards, cyano group and NaN are made₃Carry out 1,3- Dipolar Cycloaddition, and make C-5 ' position set up out desired tetrazolium part⁴¹, then enter And remove 2 ', 3 '-isopropylidene group under sour environment, you can obtain compound 16.

N⁶- and C⁵The biological assessment of the adenosine derivative of '-modify

Via MDS pharmacology service centre (MDS Pharma Services), binding ability test is demarcated with radioactivity The pharmacological property of the obtained neplanocin of (radioligand binding assay) detection.Partly representationalization Binding constant (the K of compound_i) it is listed in table 1.The A of tool potentiality_2AR excitant, CGS display lacks the activity of suppression ENT1, and ENT1 Inhibitor NBTI does not then have and A_2AThe ability that R combines.CGS and NBTI is neither difunctional medicine.The adenosine being partly obtained is similar to Thing presents to A_2AThe double activity of R and ENT1, especially, compound 1,4 and 6 is respectively for A_2AR and ENT1 has and rubs in humble That concentration and the K less than micro-molar concentration_iValue.Except compound 11 and 15, C-5 ' although the modified adenosine derivative in position Still possess the high-affinity to ENT1, but it is incorporated into A_2AThe ability of R is all destroyed.

Table 1：N⁶- and C⁵The adenosine derivative of '-the modify binding activity sub with adenosine receptor and transhipment^a.

^aIt is to be entered by MDS pharmacology service centre (Taibei, Taiwan) establishing criteria program that this radioactivity demarcates binding ability test OK.^bHuman adenosine A2A acceptor.C guinea pig balanced type transhipment 1.

We are previously mentioned, separate the compound 1 obtaining can suppress JNK activity from the methanol aqueous solution extract of rhizoma Gastrodiae And avoid the Apoptosis machine of the PC12 of serum deprivation culture to turn¹⁵.In this research, the PC12 cell of serum deprivation culture is to mark The dosage showing was through this compound treatment 24 hours.Then cell survival amount is monitored with MTT determination method, and with respect to being incubated at blood The percentage of the survival volume of clear control group is presenting.According to MTT determination method, under 0.01 μM of concentration, compound 4 and 6 is extensive The ability that the Apoptosis machine that multiple PC12 cell induces because of serum deprivation turns is identical with compound 1.In summary, these chemical combination Thing in adenosine receptors and suppresses the sub difunctional valid density that may synergistically lift adenosine of adenosine transport, especially When this two protein are close.

The statistical estimation of pharmacophore model

Fig. 4 shows A_2AThe pK of the anti-depressant pharmacophore model of R_iExperiment value is with respect to pK_iThe scatter diagram of theoretical value.pK_iReal Test value with respect to pK_iThe r of theoretical value²It is worth for 0.962, and root-mean-square error (root-mean-square of error, RMSE) For 0.658kcal/mol.This pharmacophore model further via CatScramble module, and with Fei Sheer accidental sampling (Fisher ' s randomization test) tests whether have statistical significance.This CatScramble module randomly selects this pK_iValue 19 times, to set up several new hypothesis (that is, pharmacophore model).The sampled data tool of no one of 19 hypothesis There is the COST value less than above-mentioned hypothesis.Table 2 incorporates the feature that the compound shown in Fig. 4 coincide, and assumes kiss on this compound Close the region of this feature, with the distance between the center of this feature in this pharmacophore model deviation.Come from another point of view See, table 2 is the quantization table of Fig. 4.When a part and a pharmacophoric group coincide, the degree that it coincide (or corresponding) just " is kissed with one Conjunction value " is representing.Higher identical value represents higher degree of agreement, and computer is to calculate this according to two parameters to coincide Value：The importance of this medicine bolus feature, and the accurate location of the feature on this molecule and the feature in this pharmacophore model Between proximity.

Table 2：With A_2AThe feature of the anti-depressant pharmacophore model of-R is coincide the activity of value comparative compound.Numeral in table Unit is

The A of tool potentiality_2AR excitant, CGS, all identical with four features of the pharmacophoric group of construction, and its identical feature On region distance pharmacophore model, the deviation of this feature accurate location is also very little.Compared to CGS, compound 1 shortcoming one The hydrophobic parts that the individual feature with this pharmacophore model is coincide, therefore assume the affinity of reduction.And NBTI then lacks one Individual aromatic moiety, and therefore lose and excite adenosine A_2AThe activity of acceptor.This is shown in this pharmacophore model, fragrance The possible relatively hydrophobic property part of ring feature is more important.Compound 6 misfits the hydrophobic character in pharmacophore model, and compound 11 present higher identical value.However, in comparison, the result of above-mentioned binding ability test then points out compound 6 (K_i =4.39 μM) present higher than compound 11 (K_i=41.8 μM) about ten times of binding affinity.This can also reasonable dismissal be It is because that the deviation of the position of all features and this feature accurate location on pharmacophoric group on compound 6 is less.

The pharmacophore model of the hENT1 inhibitor of this foundation only has three features, is an aromatic rings feature respectively And two hydrogen bond receptors.pK_iExperiment value is with respect to pK_iThe r of theoretical value²It is worth for 0.927, and RMSE is 0.85kcal/mol (figure 5).This drug effect graph model is further through CatScrambler module testing.This five compound (NBTI, 1, CGS, 6 and 11) is all These three features can be fit to, the therefore deviation apart from the accurate location of this feature needs to be compared (table 3).It is apparent that most The inhibitor of potentiality, NBTI, there is the identical value of highest, and three features are all had to the deviation of minimum.Compound 1,11 It is respectively 6.61,6.4 and 5.86 with 6 identical value, the active sequence consensus measuring with it.CGS (has high value of coincideing：7.1) Due to there is no inhibitory activity to hENT1, it is apparent that being excluded outside this model.However, CGS is that uniquely have to be able to It is fit to the compound (Fig. 3) of the aromatic rings feature of nucleoside moiety.Therefore, whether true modestly inspect this identical functional group The functional group that reality is same as defining this character pair on the compound of the test data set of pharmacophore analysis is extremely important 's.Only " value of coincideing " can not be used as the consideration of well-formedness.

Table 3：It is coincide with the feature of the pharmacophore model of ENT1 inhibitor the activity of value comparative compound.Numeral in table Unit is

Conclusion

We to be designed by the way of double pharmacophore models and to be incorporated into A_2AThe difunctional chemical combination of R massage transfer system Thing.Based on the structural framework of compound 1, we have designed and synthesized a series of adenosine derivative, and, if pharmacophoric group Compound after prediction is modified has acceptable activity, is just chemically modified for adenosine.This Ligand Competition binding tests Result confirm design compound really all can be suitable affinity and A_2AR and ENT1 combines.According to representative drugs In the oral dose of mouse, the compound of this design is used for treating neurodegenerative disease (inclusion Huntington's disease) T1-11 Effective dose is 1.5-2.5mg/kg.Additionally, whether speed puts formulation or sustained release forms, the compound of this design is preferred Method of administration is all oral.In summary, these compounds verified have avoid the apoptotic of serum deprivation PC12 cell Ability, and these feature critical point out its have treatment neurodegenerative disease potentiality.

Experimental design

Materials and methods

The reagent of all uses and solvent are all reagent grade (reagent grade), and, unless specifically stated otherwise, no Then do not need extra purification step before use.Oxolane (tetrahydrofuran) and diethyl ether (diethyl Ether it is) to be obtained with sodium/diphenylketone (Na/benzophenone) distillation.CH₂Cl₂It is with CaH₂Distill and obtain.All to sky The experiment of gas or moisture-sensitive is carried out all in argon gas.All glass apparatus in using front prior to baking oven in dry little more than two When, it is cooled to room temperature in the dry device of rush (desiccators).Microwave reaction is to focus on single mold microwave instrument (focused Single mode microwave unit) (CEM Discover) carried out.This instrument is provided with the microwave conveying of a sequential focusing Device, this device is simultaneously provided with a selecting type power output unit.

Fusing point is to be recorded by melting point detector (Yanaco micro apparatus).Optical activity is with digital polariscopy Instrument measures (Japan JASCO Co.DIP-1000).[α]_DThe unit of value is 10^–1deg cm²g^–1.Infrared spectrum (IR) be with Infrared spectrometer (Nicolet Magna 550-II) records.NMR spectra is through NMR (Varian Unity Plus-400) (400MHz) records, additionally, chemical deviation (chemical shifts, δ) corresponds to CHCl₃/CDCl₃δ_H 7.24/δ_C77.0 (the central spectral lines of t), (CH₃)₂CO/(CD₃)₂The δ of CO_H2.05/δ_C29.92、CH₃OH/CD₃The δ of OD_H 3.31/δ_C49.0 and (CH₃)₂SO/(CD₃)₂The δ of SO_H2.49(m)/δ_C39.5 (m), and to be represented with ppm.This heading signal (splitting patterns) be then shown as unimodal (singlet, s), doublet (doublet, d), triplet (triplet, T), quartet (quartet, q), multiplet (multiplet, m) and broad peak (broad, br).Coupling constant (coupling Constants, J) it is in units of Hz.It is with high parsing mass spectrograph (Bruker that EFI spills mass spectrum (ESI-MS) experiment Daltonics BioTOF III) carry out.Thin layer chromatography analysis test (Analytical thin-layer Chromatography, TLC) it is to carry out in thin layer chromatography board (E.Merck silica gel 60F254plates, 0.25mm), and change Compound is to be shown with ultraviolet light, anisaldehyde (anisaldehyde) spray or ninhydrin (ninhydrin) spray.Tubing string Chromatography is the tubing string of the silica gel to be filled with 70 230 mesh carrying out.It is used in A_2A- R and ENT1 test compound be through HPLC purifies and measures its purity under the absorbing wavelength of 280nm is >=95%.

The foundation of pharmacophore model

UseCompany is gone out'sModule is setting up mankind A_2AR excitant and The pharmacophore model of mankind ENT 1 inhibitor.Chemical constitution with regard to the compound of test data set and its with mankind A_2AR The information of the binding affinity of (or mankind ENT 1), be from document collect and obtain^41-44.These are used for setting up pharmacophoric group The activity of compound must cover at least four orders of magnitude, and the compound numbers in each Logarithmic degree at least will have three Individual^23,45.We also advise, such as this test data set, select the compound with big chemical variability⁴⁵.Each is changed Compound is to draw out sketch with chemical drawing software (ChemDraw) in advance, then inputs in Catalyst 4.11, then utilizes " Best " option is producing configuration.?In software, had in low-energy structure from all using polling algorithm To 250 energy not higher than structures of the lowest energy structure 20.0kcal/mol.Have selected five characterization of molecules, be respectively：Dredge Aqueous (HPh), hydrogen bond receptor (HBA), hydrogen-bond donor (HBD), cation atom (positively ionizable atom, PI), anion atom (negatively ionizable atom, NI).All these compound all can load The electrical form (spreadsheet) of catalyst software, and set default inaccuracy as 3.Other setup parameters are then Maintain default value.

N⁶- (4- hydroxy benzenes) adenosine (1)⁹

By hydrochloric acid azanol (hydroxylamine hydrochloride, 1.29g, 18.6mmol) and sodium acetate (sodium acetate, 1.67g, 20.4mmol) adds the ethanol solution of 4- hydroxy benzaldehyde (1.25g, 10.2mmol) (20mL).Stir 6 hours of mixture of this reaction at room temperature.Then ethanol is made to exclude by reduction pressure.Then, by water Add in remnant, and with Et₂O (3 ×) extracts.By the organic layer of gained with MgSO₄It is dried.With rotary evaporator under decompression After (rotary evaporation) makes volatile substance remove, make remnant in CH₂Cl₂Middle recrystallization, just can obtain 4- hydroxy benzenes first The oxime derivatives (1.3g, 93%) of aldehyde, C₇H₇NO₂, it is faint yellow solid, and fusing point (mp) is 92.0-93.6 DEG C.

At atmosheric pressure and in the presence of 10%Pd/C (80mg), make one contain above-mentioned oxime derivatives solution (342mg, 2.5mmol) and a concentration hydrochloric acid (1mL) in ethanol carries out the hydrogenization (hydrogenation) of 4 hours.Make Reactant mixture filters through silicoglaserite (Celite).Filter liquor is concentrated and is derived with the hydrogen chloride salt of output 4-Hydrobenzylamine Thing, it is faint yellow solid, and before for next step, is not required to additional purification again.

By 4-Hydrobenzylamine (395mg, in the form of this hydrogen chloride salt), 6-chlro-purine-riboside (6-chloropurine Riboside, 143mg, 0.5mmol) and the diisopropylethylamine (DIEA, 2mL, 12mmol) in 1- propyl alcohol be mixed into one mix Compound, and this mixture is heated 6 hours in 70 DEG C.Then, after reduction pressure makes this mixture concentrate, add water development shape Become white precipitate.This white precipitate just can get the compound 1 (151mg, 81%) described in title after filtering.Through carrying The purity that the HPLC of Inertsil ODS-3 tubing string (4.6 × 250mm, 5 μm) measures this product is>99%.Additionally, this product (C₁₇H₁₉N₅O₅) it is white powder, fusing point is 208.7-209.2 DEG C of (document⁹It is recited as 216-219 DEG C)；[α]²⁰ _D=-64.5 (DMSO, c=1) (document⁹Record [α]²⁵ _D=-87 (MeOH, c=0.1))；TLC(MeOH/EtOAc(1:9))R_f=0.3；¹H NMR(DMSO-d₆, 400MHz) δ 9.22 (1H, s), 8.34 (1H, s), 8.30 (1H, br s), 8.18 (1H, s), 7.12 (2H, d, J=8.0Hz), 6.65 (2H, d, J=8.0Hz), 5.86 (1H, d, J=5.6Hz), 5.41 (2H, m), 5.18 (1H, d, J= 5.6Hz), 4.60 (2H, m), 4.13 (1H, q, J=4.6,7.4Hz), 3.95 (1H, q, J=3.4,6.2Hz), 3.66 (1H, M), 3.53 (1H, m)；¹³C NMR(DMSO-d₆, 400MHz) and δ 155.3,153.6,151.6,147.6,139.1,129.5, 127.9 (2 ×), 119.2,114.4 (2 ×), 87.6,85.6,73.3,70.5,61.5,42.4；Matter lotus is calculated with ESI-HRMS Than for 374.1412 [M+H]⁺(C₁₇H₂₀N₅O₅Theoretical value：374.1459).

N⁶- (3- indolylethyl) adenosine (6)

By a 6-chloropurine deoxyribonucleoside (6-chloropurine ribonucleoside) solution (71mg, 0.25mmol), the tryptamines (tryptamine, 101mg, 0.63mmol) and in ethanol (3mL) and diisopropylethylamine (0.24mL, 2.88mmol) is placed in a round-bottomed flask.This flask is placed in the groove room of a focusing single mold microwave instrument, and with The intensity illumination of 150W processes 10 minutes so that alcohol reflux (refluxing).Then this mixture is concentrated with rotary evaporator, And by fast layer analyzer (flash chromatography；Silica gel, MeOH/EtOAc (1：9)) purify remnant, you can obtain Obtain compound 6 (85mg, 83%).(liquid phase adopts HPLC through carrying HC-C18 tubing string (Agilent, 4.6 × 250mm, 5 μm) The molten CH from gradient for 30-60%₃The CN aqueous solution) purity that measures this product is>99%.Additionally, this product (C₂₀H₂₂N₆O₄) For white powder, fusing point is 187.0-187.2 DEG C；[α]²⁰ _D=-55.7 (CH₃OH, c=1.0)；TLC(MeOH/EtOAc(1： 9))R_f=0.41；¹H NMR(DMSO-d₆, 400MHz) δ 10.78 (1H, s), 8.33 (1H, s), 8.25 (1H, s), 7.96 (1H, Br s), 7.61 (1H, d, J=7.2Hz), 7.32 (1H, d, J=9.2Hz), 7.18 (1H, s), 7.05 (1H, t, J=8.0Hz), 5.80 (1H, d, J=6.0Hz), 5.47-5.44 (2H, m), 5.20 (1H, d, J=4.4Hz), 4.61 (1H, d, J=5.6Hz), 4.14 (1H, d, J=2.8Hz), 3.96 (1H, d, J=3.2Hz), and 3.77 (1H, br s), 3.69-3.52 (2H, m), 3.01 (2H, t, J=7.2Hz)；¹³C NMR(DMSO-d₆, 100MHz) and δ 154.3,152.2,148.0,139.5,136.0,127.1, 122.4,120.8,119.6,118.3,118.1,111.7,111.2,87.9,85.8,73.4,70.6,61.6,40.5,25.1； Mass-to-charge ratio is calculated for 411.1750 [M+H] with ESI-HRMS⁺(C₂₀H₂₃N₆O₄Theoretical value：411.1775).

5 '-repeatedly nitrogen base -5 '-deoxidation -2 ', 3 '-O- isopropylidene-N⁶- (4- methoxybenzyl) adenosine (the third of compound 8 Ketone acetalation derivative)

To be dropwisely added in the paratoluensulfonyl chloride (6.3g, 34.6mmol) in anhydrous pyridine (6.0mL) with syringe The N of the cyclic acetal in anhydrous pyridine (36mL)⁶- (4- methoxybenzyl) adenosine (compound 3 of cyclic acetal, 2.96g, 6.9mmol) in.Stir 6 hours of this mixture at room temperature.Then pyridine is made to remove by decompression, then with CH₂Cl₂ And H₂O extracts remnant.Then, make organic layer through MgSO₄Be dried, through filter, concentrated again and obtain monosulfonate derivative and The mixture (5 of chlorine derivative：1), as¹Shown in H NMR spectra.

Above-mentioned prepared mixture is dissolved in dry DMF (70mL), and add sodium azide (1.34g, 20.6mmol).Again this mixture is stirred 6 hours at 80 DEG C, then reduce pressure to be concentrated.Then, with CH₂Cl₂With H₂O extracts remnant, then makes organic layer through MgSO₄Be dried, through filter, more concentrated to obtain a pale yellow oil.This is yellowish Color grease is further through flash chromatography method (silica gel；CH₂Cl₂/MeOH(100：1)) purify and obtain the acetone acetal of compound 8 Change derivative (8-acetonide, 653mg, gross production rate：21%).This product (C₂₁H₂₄N₈O₄) it is colorless oil；TLC (EtOAc/Hexane(6:4))R_f=0.39；[α]²³ _D=+5.0 (EtOAc, c=1.0)；IR ν_max(neat) 3280,2987, 2931,2101,1618,1512,1478,1375,1330,1296,1248,1211,1154,1091cm^–1；¹H NMR(CDCl₃, 400MHz) δ 8.38 (1H, br s), 7.72 (1H, br s), 7.26 (2H, d, J=8.8Hz), 6.84 (2H, d, J=8.8Hz), 6.37 (1H, br s), and 6.06 (1H, d, J=2.0Hz), 5.46-5.44 (1H, m), 5.07-5.05 (1H, m), 4.77 (2H, br S), 4.38-4.35 (1H, m), 3.77 (3H, s), 3.51-3.62 (2H, m), 1.61 (3H, s), 1.39 (3H, s)；¹³C NMR (CDCl₃, 100MHz) and δ 158.7,154.5,153.1,147.9,139.0,130.2,128.8 (2 ×), 120.1,114.4, 113.8 (2 ×), 90.4,85.6,83.9,82.0,55.2,52.2,43.7,29.7,27.1,25.3；Matter is calculated with ESI-HRMS Lotus is than for 453.1999 [M+H]⁺(C₂₁H₂₄N₈O₄Theoretical value：453.1999).

5 '-acetamide -5 '-'-deoxy-n⁶- (4- methoxybenzyl) adenosine (9)

At room temperature, make acetone acetalation derivative (95mg, 0.21mmol) of compound 8 and the triphenyl phasphine containing repeatedly nitrogen base (triphenylphosphine, 66mg, 0.24mmol) is in THF/H₂O(10：1,2mL) 4.5 hours of stirring in.Then, subtract Pressure makes this mixture concentrate.With CH₂Cl₂And H₂O extracts (taken up) remnant, then is allowed to be acidified with hydrogen chloride solution (HCl) To pH=2.Then, water layer is separated, with saturation NaHCO₃In the aqueous solution and after, then with CH₂Cl₂Extracted.Then, made Machine extract is through MgSO₄Be dried, through filter, more concentrated to obtain a rough amine product.

Make the reaction in anhydrous pyridine (0.2mL) of this rough amine product and acetic anhydride (98.6 μ L, 1.05mmol).In room Temperature is lower to stir 1.5 hours of this mixture, then reduces pressure and so that it is concentrated.Then, with CH₂Cl₂And H₂O extracts remnant, then makes organic Layer is through MgSO₄Be dried, through filter, concentrated, again through flash chromatography method (silica gel；CH₂Cl₂/MeOH(98：2)) purify, and obtain The acetone acetalation derivative (56mg, the yield of two steps is 57%) of compound 9.Through carry HC-C18 tubing string (Agilent, 4.6 × 250mm, 5 μm) HPLC (liquid phase adopts the molten CH from gradient for 30-60%₃The CN aqueous solution) measure the purity of this product For>99%.This product (C₂₃H₂₈N₆O₅) it is a colorless oil；TLC(CH₂Cl₂/MeOH(98：2))R_f=0.2；[α]²⁵ _D= 146.6(CHCl₃, c=1.0)；IR ν_max(neat) 3280,3062,2989,2930,2835,2358,1667,1620,1513, 1376,1336,1296,1246,1215,1096,1034cm^–1；¹H NMR(CDCl₃, 400MHz) δ 8.36-8.38 (2H, m), 7.73 (1H, s), 7.28 (2H, d, J=8.8Hz), 6.86 (2H, d, J=8.8Hz), 6.15 (1H, br s), 5.77 (1H, d, J =4.8Hz), 5.26 (1H, t, J=4.8Hz), 4.81 (1H, dd, J=4.0,2.4Hz), 4.76 (2H, br s), 4.47-4.48 (1H, m), 4.11-4.17 (1H, m), 3.79 (3H, s), 3.24 (1H, d, J=14.4Hz), 2.15 (3H, s), 1.61 (3H, s), 1.34 (3H, s)；¹³C NMR(CDCl₃, 100MHz) and δ 170.5,158.8,154.8,152.7,147.7,139.7,130.0, 128.9 (2 ×), 121.1,114.6,113.9 (2 ×), 92.5,83.3,82.2,81.3,55.3,43.9,41.1,27.6, 25.4,23.2；Mass-to-charge ratio is calculated for 469.2193 [M+H] with ESI-HRMS⁺(C₂₃H₂₈N₆O₅Theoretical value：469.2190).

Acetone acetalation derivative (17.2mg, 0.037mmol) making this compound 9 at room temperature is in 3M HCl/THF (1:1,0.1mL) 14 hours of stirring in, then again with saturation NaHCO₃The aqueous solution neutralizes.Then decompression makes this mixture dense Contracting, then with CH₂Cl₂And H₂O extracts remnant.Then, make organic layer through MgSO₄Be dried, through filtering, concentrated again and obtain mark Compound 9 (11mg, 70%) shown in topic.Through carrying the HPLC (liquid of HC-C18 tubing string (Agilent, 4.6 × 250mm, 5 μm) Mutually adopt the molten CH from gradient for 30-60% in 20 minutes₃The CN aqueous solution) purity that measures this product is>99%.This product (C₂₀H₂₄N₆O₅) it is white powder；Fusing point is 121.1-121.6 DEG C；TLC(CH₂C_l2/MeOH(9：1))R_f=0.5；[α]²⁵ _D=- 108.7 (THF, c=0.89)；IR ν_max(neat) 3275,3071,2923,2852,2360,1621,1512,1375,1339, 1297,1245,1175,1126,1076cm^–1；¹H NMR(CDCl₃, 400MHz) δ 8.76 (1H, s), 8.27 (1H, s), 7.24 (2H, d, J=8.4Hz), 6.81 (2H, d, J=8.4Hz), 6.54 (1H, s), 5.70 (1H, d, J=5.6Hz), 4.72 (3H, d, J=5.6Hz), 4.23 (1H, s), 4.18 (1H, s), 3.98-4.03 (1H, m), 3.75 (3H, s), 3.13 (1H, d, J= 14.0Hz), 2.02 (3H, s)；¹³C NMR (DMSO, 100MHz) δ 169.4,157.9,154.3,152.3,148.3,140.2, 131.8,128.4 (2 ×), 119.8,113.5 (2 ×), 87.9,83.6,72.6,71.3,55.1,42.4,41.1,22.7；With It is 427.1727 [M+H] that ESI-HRMS calculates mass-to-charge ratio⁺(C₂₀H₂₄N₆O₅Theoretical value：427.1730).

5 '-deoxidation -5 '-(4- methyl isophthalic acid, 2,3- triazol-1-yl)-N⁶- (4- methoxybenzyl) adenosine (11)

At room temperature by acetone acetalation derivative (313mg, 0.69mmol) of the compound 8 containing repeatedly nitrogen base, CuSO₄· 5H₂O (24.9mg), sodium ascorbate (sodium ascorbate, 61.4mg) and propargyl alcohol are in H₂O/t-BuOH(1:1,7mL) To become a mixture, then decompression makes this mixture concentrate to 12 hours of middle stirring.Then, with CH₂Cl₂And H₂O extraction remaining Thing.Then, make organic layer through MgSO₄Be dried, through filtering, concentrated again and triazole compounds of output one acetone acetalation (triazole acetonide ,～220mg), it is a colorless oil；TLC(CH₂Cl₂/MeOH(9：1))R_f=0.5；With It is 509.2267 [M+H] that ESI-HRMS calculates mass-to-charge ratio⁺(C₂₄H₂₈N₈O₅Theoretical value：509.2261).

At room temperature by the triazole compounds being above obtained and triethylamine (triethylamine, 0.15mL, 1.08mmol) With mesyl chloride (methylsulfonyl chloride, 0.08mL, 1.08mmol) in anhydrous CH₂Cl₂(4.3mL) stirring two in Individual hour.Then decompression makes this mixture concentrate, then with CH₂Cl₂And H₂O extracts remnant.Then, make organic layer through MgSO₄Dry Dry, through filtering, concentrated again and output one mesylate compound (C₂₅H₃₀N₈O₇SNa), it is colorless oil；TLC(EtOAc/ Hex(4：1))R_f=0.45；Mass-to-charge ratio is calculated for 609.1876 [M+H] with ESI-HRMS⁺(C₂₅H₃₀N₈O₇SNa theoretical value： 609.1856).

Above-mentioned mesylate compound and NaBH is made at 0 DEG C₄(24.5mg, 0.65mmol) reacts in DMF, then in 60 Heat 6 hours at DEG C.Then decompression makes this mixture concentrate, then with CH₂Cl₂And H₂O extracts remnant.Then, make organic layer Through MgSO₄Be dried, through filtering, the concentrated again and acetone acetalation derivative of output one compound 11, it is colorless oil； TLC(EtOAc/Hex(4:1))R_f=0.25；Mass-to-charge ratio is calculated for 493.2312 [M+H] with ESI-HRMS⁺(C₂₄H₂₈N₈O₄Theoretical It is worth and be：493.2312).

At room temperature by the acetone acetalation derivative of above-claimed cpd 11 in the HCl/THF (1 of 3M:1,0.33mL) stir in Mix 14 hours, then with saturation NaHCO₃The aqueous solution is neutralized.Then decompression makes this mixture concentrate, then remnant is molten Solution, after THF, obtains the compound 11 (48.1mg, overall productivity is 25%) shown in title through filtering, concentrating.Through carrying (liquid phase adopts the molten CH from gradient for 30-60% to the HPLC of HC-C18 tubing string (Agilent, 4.6 × 250mm, 5 μm)₃CN is water-soluble Liquid) measure this product purity be 98%.This product (C₂₁H₂₄N₈O₄) it is white powder；Fusing point is 183.0-183.2 DEG C；TLC (CH₂Cl₂/MeOH(9:1))R_f=0.12；[α]²⁷ _D=+20.3 (CH₃OH, c=0.45)；IR ν_max(neat) 3217,2921, 2850,2685,1620,1513,1470,1337,1297,1244,1176,1111,1058cm^-1；¹H NMR(CD₃OD, 400MHz) δ 8.22 (1H, s), 7.99 (1H, s), 7.45 (1H, s), 7.31 (2H, d, J=8.8Hz), 6.87 (2H, d, J= 8.8Hz), 5.96 (1H, d, J=4.0Hz), 4.82-4.68 (5H, m), 4.46 (1H, t, J=4.0Hz), 4.34 (1H, q, J= 4.0Hz), 3.77 (3H, s), 2.15 (3H, s)；¹³C NMR(CDCl₃, 100MHz) and δ 158.7,154.2,152.9,148.0, 142.8,138.8,130.1,128.8 (2 ×), 123.1,119.6,113.8 (2 ×), 89.3,82.2,73.4,70.8,55.2, 50.9,43.9,10.5；Mass-to-charge ratio is calculated for 451.1843 [M-H] with ESI-HRMS (anion scanning)^-(C₂₁H₂₄N₈O₄Theoretical It is worth and be：451.1842).

3,4- dihydroxy -5- [6- (4- hydroxyphenylmethyl amido)-purine -9- base]-oxolane -2- carboxylic acyloxy ethamine (15)

At 40 DEG C, make acetone acetalation derivative (the acetone acetalation of compound 17 of 6-chloropurine core furanoside Derivative, 158mg, 0.48mmol) and PhI (OAc)₂(509mg, 1.56mmol) and 2,2,6,6- tetramethyl piperidine -1- epoxides (TEMPO, 15.4mg, 0.1mmol) is in the CH of degasification (degassed)₃CN/H₂O solution (1:1,2.6mL) 4 hours of stirring in. Then decompression makes this mixture concentrate and the rough acid compound of output 18.

At room temperature, this rough acid compound and ethamine (117mg, in the form of hydrogen chloride salt), o- (benzo are made Triazol-1-yl)-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid (O- (benzotriazol-1-yl)-N, N, N ', N '- tetramethyluronium hexafluorophosphate；HBTU, 375mg, 0.72mmol) and diisopropylethylamine (0.5ml, 2.89mmol) 14 hours of reaction in dry DMF (11.6mL).Then, after decompression makes this mixture concentrate, with CH₂Cl₂And H₂O extracts remnant, and makes organic layer through MgSO₄Be dried, through filter, concentrated, again through flash chromatography method (silica gel； EtOAc/hexane(1：1)) purify, just obtain the amide product 19 of a colorless oil.

Then, at 70 DEG C, (385mg, 2.4mmol, with the shape of hydrogen chloride salt with 4-Hydrobenzylamine to make this amide product Formula) and diisopropylethylamine (2.8mL, 16.9mmol) 2 hours of reaction in 1- propyl alcohol (28mL).Then, decompression makes this mix After compound concentrates, with CH₂Cl₂And H₂O extracts remnant, and makes organic layer through MgSO₄Be dried, through filtering, concentrated, again through fast Fast chromatography (silica gel；CH₂Cl₂/MeOH(97：3)) purify, and obtain compound 15 acetone acetalation derivative (179mg, 82%).This product (C₂₃H₂₈N₆O₅) it is colorless oil；TLC(EtOAc/hexane(4：1))R_f=0.13；[α]²² _D=- 32.0 (EtOAc, c=1.0)；IR ν_max(neat) 3347,3103,2982,2933,1732,1667,1615,1516,1479, 1461,1376,1332,1295,1245,1212,1154,1088cm^-1；¹H NMR(CDCl₃, 400MHz) and δ 9.01 (1H, br S), 8.35 (1H, br s), 7.81 (1H, s), 7.14 (1H, br s), 7.04 (2H, d, J=8.0Hz), 6.68 (2H, d, J= 8.0Hz), 6.49 (1H, t, 4.8Hz), 6.03 (1H, d, J=2.4Hz), 5.33-5.38 (2H, m), 4.70 (3H, s), 3.09- 3.16 (2H, m), 1.62 (3H, s), 1.37 (3H, s), 0.90 (3H, t, J=7.2Hz)；¹³C NMR(CDCl₃, 100MHz) and δ 168.7,155.8,154.2,153.1,147.7,139.1,128.8 (3 ×), 119.6,115.4 (2 ×), 114.3,91.6, 85.6,83.3,82.4,43.9,34.0,27.0,25.1,14.2；Mass-to-charge ratio is calculated for 455.2037 [M+H] with ESI-HRMS⁺ (C₂₂H₂₆N₆O₅Theoretical value：455.2043).

At room temperature, acetone acetalation derivative (26mg, 0.057mmol) making this compound 15 is in 1M HCl/THF (1:1,0.3mL) 16 hours of stirring in, then with the NaHCO of saturation₃The aqueous solution is neutralized.After concentrating, make this remaining Thing and water development (triturated) and obtain the compound 15 shown in title.This compound 15 then through MeOH (14.65mg, 62%) recrystallize.This product (C₁₉H₂₂N₆O₅) it is white powder, fusing point is 179.7-180.5 DEG C；TLC(EtOAc)R_f= 0.04；[α]²³ _D=-27.7 (MeOH, c=1.0)；IR ν_max(neat) 3256,2688,2360,1618,1515,1335, 1294,1232,1128,1052cm^-1；¹H NMR(CD₃OD, 400MHz) δ 8.29 (1H, s), 8.22 (1H, s), 7.20 (2H, d, J =8.4Hz), 6.73 (2H, d, J=8.4Hz), 6.00 (1H, d, J=7.6Hz), 4.76-4.73 (1H, m), 4.70 (2H, br S), 4.46 (1H, s), 4.30-4.31 (1H, m), 3.36 (2H, q, 7.2Hz), 1.21 (3H, t, 7.2Hz)；¹³C NMR(CD₃OD, 100MHz) δ 171.8,157.5,155.8,153.6,149.1,141.9,130.5,129.9 (2 ×), 121.3,116.2 (2 ×), 90.5,86.3,74.9,73.4,44.9,35.2,15.3；Mass-to-charge ratio is calculated for 413.1573 [M-H] with ESI-HRMS⁺ (C₁₉H₂₁N₆O₅Theoretical value：413.1573)；The result of elementary analysis is：C, 52.88；H, 5.40；N, 19.44 (C₁₉H₂₂N₆O₅·H₂O theoretical value：C, 52.77；H, 5.59；N, 19.43).

2- [6- (4- methoxybenzyl amido)-purine -9- base] -5- (1H-TETRAZOLE -5- base)-oxolane -3,4- glycol (16)

At 50 DEG C, by acetone acetalation derivative (ca.3.98mmol) of compound 17 through PhI (OAc)₂/ TEMPO oxygen Rough acid compound 18 obtained by change in dry DMF (in (40mL) with ammonium chloride (426mg, 7.96mmol), BTA- 1- base-epoxide tripyrrole alkyl phosphorus hexafluorophosphoric acid (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-fluorophosphate；PyBOP, 3.07g, 5.97mmol), hydroxyl BTA (hydroxybenzotriazole, HOBt, 807mg, 5.97mmol) and diisopropylethylamine (2.5mL, 15.9mmol) 14 hours of reaction.Then, decompression makes this After mixture concentrates, with CH₂Cl₂And H₂O extracts remnant, and makes organic layer through MgSO₄Be dried, through filtering, concentrated, warp again Flash chromatography method (silica gel；EtOAc/hexane(1：1 to 4：1)) purify, and obtain the amide product 20 of a colorless oil.

At -78 DEG C, one is dissolved in CH₂Cl₂(10mL) dimethyl sulphoxide solution (0.85mL, 11.9mmol) adds one It is dissolved in CH₂Cl₂(10mL) in oxalyl chloride solution (0.7mL, 7.96mmol).Stir this mixture 30 minutes, be subsequently adding one molten In CH₂Cl₂(20mL) amide compound 20 (ca.3.98mmol).Stir this mixture 30 minutes at -78 DEG C, Ran Houjia Enter diisopropylethylamine (2.6mL, 15.9mmol).After 1 hour of stirring, go out nitrile compounds via TLC testing inspection 21 formation.With CH₂Cl₂And H₂O extracts this mixture, and makes organic layer through MgSO₄Be dried, through filtering, concentrated, again through fast Fast chromatography (silica gel；EtOAc/hexane(2：3)) purify, and the nitrile compounds 21 (863mg) of output one colorless oil, should Nitrile compounds 21 are simultaneously contaminated with a little HOBt.

At 70 DEG C, by above-mentioned prepared nitrile compounds (863mg, 2.68mmol) and 4- methoxybenzyl amine (1.84g, 13.4mmol) and diisopropylethylamine (15.5mL) 4 hours of reaction in 1- propyl alcohol (26mL).Then, decompression makes After this mixture concentrates, with CH₂Cl₂And H₂O extracts remnant, and makes organic layer through MgSO₄Be dried, through filtering, concentrated, again Through flash chromatography method (silica gel；CH₂Cl₂/MeOH(300：1 to 150：1)) purify, and obtain compound 22 (905mg, overall productivity For 54%).This product (C₂₁H₂₂N₆O₄) it is colorless oil；TLC(EtOAc/hexane(4：1))R_f=0.55；[α]²⁶ _D=+ 25.8 (EtOAc, c=1.0)；IR ν_max(neat) 3373,3282,2990,2925,2853,1679,1618,1512,1465, 1376,1331,1295,1249,1212,1135,1086cm^-1；¹H NMR(CDCl₃, 400MHz) and δ 8.39 (1H, br s), 7.64 (1H, br s), 7.26 (2H, d, J=10.4Hz), 6.83 (2H, d, J=10.4Hz), 6.54 (1H, t, J=5.6Hz), 6.13 (1H, s), 5.77 (1H, d, J=4.0Hz), 5.68 (1H, dd, J=1.6,4.0Hz), 4.95 (1H, d, J=1.6Hz), 4.75 (2H, br s), 3.79 (3H, s), 1.57 (3H, s), 1.42 (3H, s)；¹³C NMR(CDCl₃, 100MHz) and δ 158.8, 154.5,153.2,148.2,138.9,130.2,128.9 (2 ×), 119.7,115.9,114.5,113.9 (2 ×), 91.6, 84.6,83.9,75.1,55.3,44.0,26.6,25.1；Mass-to-charge ratio is calculated for 421.1612 with ESI-HRMS (anion scanning) [M-H]^-(C₂₁H₂₂N₆O₄Theoretical value：421.1624).

Will be in the nitrile compounds 22 solution (905mg, 2.14mmol) in DMF (20mL) and NH₄Cl (429mg, After 8.04mmol) being cooled to 0 DEG C, add NaN₃(523mg, 8.04mmol).Then, ice bath is removed, make this mixture in 40 DEG C 1 hour of heating, then lentamente heats to 90 DEG C again, and is maintained at 90 DEG C and stirs 9 hours of this mixture.Then, This mixture is cooled down, and is concentrated through by decompression.Then so as to after being dissolved in EtOAc, through NaHCO₃The aqueous solution (pH= 8) extract, then add HCl solution (1M) to water layer being acidified to after pH=2, then with CH₂Cl₂Extraction.Then, make organic Layer is through MgSO₄Be dried, through filtering, concentrated again and obtain a substantially pure tetrazolium product, i.e. the third of compound 16 Ketone acetalation derivative.This product (C₂₁H₂₃N₉O₄) it is colorless oil (460mg, yield is 46%)；TLC(CH₂Cl₂/MeOH (9：1))R_f=0.25；[α]²⁷ _D=+13.2 (EtOAc, c=1.0)；IR ν_max(neat) 3361,2926,2852,1613, 1513,1481,1375,1333,1293,1249,1210,1176,1154,1101,1034cm^-1；¹H NMR(CDCl₃, 400MHz) δ 7.90 (1H, br s), 7.68 (1H, br s), 7.35 (2H, d, J=8.4Hz), 6.86 (2H, d, J=8.4Hz), 6.83 (1H, br s), 6.18 (1H, s), 5.85 (1H, s), 5.73 (1H, d, J=6.0Hz), 5.49 (1H, d, J=6.0Hz), 4.92 (1H, dd, J=6.8,7.6Hz), 4.39 (1H, dd, J=4.0,10.4Hz), 3.77 (3H, s), 1.69 (3H, s), 1.43 (3H, s)；¹³C NMR(CDCl₃, 100MHz) and δ 158.4,154.9,152.9,152.6,146.2,138.5,129.5,129.2 (2 ×), 118.4,114.2,113.7 (2 ×), 93.4,85.9,83.7,82.3,55.4,44.1,27.1,25.2；With ESI-HRMS (anion scanning) calculates mass-to-charge ratio (C₂₁H₂₃N₉O₄Theoretical value：464.1795) it is 464.1786 [M-H]^-.

Acetone acetalation derivative (460mg, 0.99mmol) making compound 16 at room temperature is in the HCl/THF (1 of 3M： 1,0.1mL) 14 hours of stirring in, then with the NaHCO of saturation₃The aqueous solution is neutralized.Then, decompression makes this mixture dense After contracting, (taken up) remnant is extracted with THF, then so that it is filtered, concentrate and obtain compound 16 shown in title (320mg, 76%).Through carry HC-C18 tubing string (Agilent, 4.6 × 250mm, 5 μm) HPLC (liquid phase adopt 20 minutes in molten from gradient CH for 30-60%₃The CN aqueous solution) measure this product purity be 99%.This product (C₁₈H₁₉N₉O₄) it is white powder；Fusing point For 210.0-210.6 DEG C；TLC(CH₂Cl₂/MeOH(9：1))R_f=0.05；[α]²⁶ _D=-25.8 (THF, c=1.0)；IR ν_max (neat) 3397,2841,2692,1623,1511,1475,1419,1339,1302,1236,1180,1124,1045cm^-1；¹H NMR(DMSO-d₆, 400MHz) δ 8.82 (1H, s), 8.30 (1H, br s), 8.20 (1H, s), 7.26 (2H, d, J=8.0Hz), 6.83 (2H, d, J=8.0Hz), 6.08 (1H, d, J=5.6Hz), 5.53 (1H, d, J=6.0Hz), 5.46 (1H, d, J= 2.8Hz), 5.18 (1H, s), 4.91 (1H, d, J=5.2Hz), 4.62 (2H, br s), 4.20 (1H, s), 3.69 (3H, d, J= 2.0Hz)；¹³C NMR(DMSO-d₆, 100MHz) and δ 159.9,157.4,153.7,151.9,138.8,131.5,127.9 (2 ×), 113.1 (2 ×), 85.9,79.1,75.4,74.3,54.6,41.9；Mass-to-charge ratio is calculated with ESI-HRMS (anion scanning) For 427.1727 [M-H]^-(C₁₈H₁₉N₉O₄Theoretical value：427.1730).

Radioactivity demarcates binding test

It is with radioactivity via MDS pharmacology service centre (MDS Pharma Services) that radioactivity demarcates binding test Demarcate binding ability test (radioligand binding assay) to carry out.In A_2AIn the binding ability test of acceptor⁴⁶, will Excessive performance mankind A_2AThe memebrane protein of the HEK293 cell of acceptor is collected in 25 DEG C contain³The reaction of H-CGS21680 (50nM) Buffer solution [50mM Tris-HCl (pH 7.4), 10mM MgCl₂, 1mM EDTA, and 2U/mL adenosine removes amine enzyme] in process 90 points Clock.Add 50 μM of adenosine -5 '-N- alkylcarboxy acid amides (NECA) to assess the situation of non-specific binding.

The binding ability test of adenosine transport is then carried out in the manner previously described⁴⁷.By duncan-Hartley guines pig (Duncan Hartley derived guinea pig) corticocerebral membranous with³The NBTI (0.5nM) that H- demarcates is altogether It is incubated at 30 minutes in 25 DEG C of the buffer solution containing 50mM Tris-HCl (pH 7.4) together.Add 5 μM of NBTI non-to assess The situation of specific binding, NBTI only can produce inhibition to ENT1 under the concentration of 0.5nM, therefore for balanced type nucleosides Sub 1 (ENT1) of transhipment is the inhibitor with high-affinity⁴⁸.This reaction can be through GF/B glass fiber filter and identical buffer solution Terminate after cleaning.

MTT metabolic analysis

Buy from the PC12 cell of ATCC (Manassas, VA, USA) be with containing 10% horse serum and 5% tire ox blood Clear DMEM nutrient solution (Dulbecco's modified Eagle's medium) maintains, and is incubated at 37 DEG C of CO₂Culture Case (5%).Survival volume is as is well known with 3- (4,5- dimethylthiazole -2- base) -2,5- diphenol tetrazolium bromide (MTT) metabolism Analysis is carried out^49,50.In brief, first by the cell being incubated in 150-mm culture plate with PBS three times, then make it again It is suspended in DMEM.Then, cell in suspension is inoculated in 96 porose discs that (each culture hole contains 1 × 10⁴Individual cell), and warp Or not designated agent treatment.After 24 hours of culture, MTT (0.5mg/mL) is added in culture medium, and is further cultured for 3 Hour.Then, after removing culture medium, the Mitochondria through living cells for the tetrazole ring in each culture hole is dissolved with DMSO and divides output Sediment (formazan crystals).Then by the micro- disk analyzer (micro-enzyme-linked of ferment immunity Immunosorbent assay (ELISA) reader) measure the light absorption value of each culture hole under wavelength 570/630nm.

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Claims (4)

1. a kind of medical composition is used for the application in the medicine treating neurodegenerative disease, wherein said medicinal combination in preparation Thing comprises a compound and a medical acceptable carrier, and wherein said compound has following structure：

Wherein

N is 1 to 3；And

The heterocycle that this is substituted contains 5 yuan or 6 yuan of rings and is somebody's turn to do and closes heterocycle and contain nitrogen, oxygen or sulphur as hetero atom, and this takes Dai Ji is selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, C_1-6The group that alkyl, trifluoromethyl and (being substituted) phenyl group are formed.

2. apply as claimed in claim 1, wherein, described compound has following structure：

3. apply as claimed in claim 1, wherein, described neurodegenerative disease is Alzheimer's disease, Parkinson's disease, flesh wither Contracting lateral schlerosis, Puli hold high disease, Huntington's disease and spinocerebellar ataxia disease or a combination thereof.

4. apply as claimed in claim 3, wherein, described neurodegenerative disease is Huntington's disease.